The proposed study concerns transcription and RNA processing in yeast mitochondria. We have previously catalogued over 40 mitochondrial RNA species other than tRNA, many of which appear to be precursors to fully processed RNA. In yeast mitochondria, processing of RNA includes excision of intervening sequences ("introns"), RNA splicing, and excision of terminal sequences. It is planned to analyze processing relations by resolving mitochondrial RNA on gels, transferring to diazobenzyloxyethyl paper, and hybridizing with specific gene probes. We will also map precursors by hybridizing to mapped restriction digests of mitochondrial DNA with an average fragment size of 400 nucleotide pairs. Once specific transcripts of a particular gene are mapped and their processing relationships defined, we will purify transcripts and analyze their translation in a reticulocyte cell-free lysate. We will also examine the effects on processing of biological manipulation of yeast mitochondria, including anaerobiasis, glucose repression, and oxygen adaptation. When we have localized promoters and processing sites in the genome, we will then define them by DNA and RNA sequencing. Further, we will attempt to characterize and purify the enzyme activities that carry out excision and splicing of introns, terminal excision of RNA segments, and tRNA processing.